The overarching goal of the proposed research is to predict the intracellular and extracellular concentration- time profiles using models that include membrane partitioning, membrane permeability, organ blood flow, active transport, and metabolism. In the funding period from 2013-2016, we have made significant progress in incorporating explicit membrane compartments into predictive models, and we have evaluated in depth the impact of various membrane geometries and related factors on intracellular concentration prediction. We are now using the basic principles underlying permeability and partitioning to build a new framework for PBPK models. This will allow us to incorporate permeability-limited distribution, partitioning, organ blood flow, and active transport into PBPK models with explicit membrane kinetics (memPBPK). This new paradigm will provide markedly better predictions of intracellular concentrations, and will address an unmet critical need for cost effective drug development by providing novel predictive tools for drug disposition in humans. Three specific aims are proposed. 1) Novel biophysical methods will be used to study the cellular kinetics of drug permeability and partitioning. Specifically, novel cell microscopy techniques will be used to evaluate the time-course of cellular distribution and conduct cellular permeability studies in monolayers, and a range of explicit membrane models developed during the current funding period will be evaluated for their ability to quantify the observed membrane, organelle, and cellular distribution kinetics. 2) Develop a new framework for PBPK and hybrid compartmental-PBPK models that incorporate membrane partitioning, permeability-limited diffusion, and organ blood flow (memPBPK). Components include organ-specific models for use in hybrid and full PBPK approaches, and models for absorption using our published convection-diffusion-reaction approach. These models will be used in Aim 3 to incorporate active uptake/efflux transport and metabolism to predict intra- and extracellular concentration-time profiles. 3) In vivo experimental data from rats and humans will be used to expand and validate models to predict the time course of intra- and extracellular drug concentrations. We will focus on modeling the disposition of drugs in the liver and the absorption of drugs from the gastrointestinal tract in the presence of transporters and drug metabolizing enzymes.